Fig 2: Enumeration of circulating DC in GBM patients and healthy controls. (a) Flow cytometry was used to enumerate circulating DC. Briefly, whole blood was stained and red blood cells lysed prior to acquisition on a flow cytometer. (i) Shows the typical forward (FSC) and side scatter (SSC) of the leukocyte populations. Doublet events were excluded by plotting FSC height against area (ii). The exclusion of B cells, monocytes and granulocytes were achieved by gating CD14-/19- and side scatter low events (iii). HLA-DR high events, a hallmark of DC, were then selected (iv). Lineage markers for cDC2 (CD1c+), pDC (CD303+) (v), cDC1(CD141high), and slanDC (Slan+) (vi) were then identified against isotype controls (purple gates). (b) Absolute numbers of circulating DC in GBM patients compared with healthy controls.(c) Correlation of steroid dose on the day of blood acquisition to DC counts (in natural log for parametric analysis). Pearson's correlation coefficient (r) and significance are shown. Dex dose is associated with lower numbers of all subsets of circulating DC * = p < 0.05.

Fig 3: Dex treatment increase the proportion of CD8+ T cells displaying an effector phenotype. (A) Representative flow cytometry data demonstrating the gating strategy used for CD8+ T cells. Forward scatter (FSC) area vs. FSC-height was used for doublet discrimination, and lymphocytes were selected by FSC vs. side scatter. A “dump” channel was then used to gate out dead cells (LIVE/DEAD fixable aqua viability stain), monocytes/macrophages (CD14), NK cells (CD56) and B cells CD19). CD8+ T cells were subsequently selected on the basis of CD8 vs. CD3 staining, followed by the identification of “effector CD8” cells as HLA-DRhiCD38hi. This population was further gated for expression of Ki67, Bcl-2 and ICOS, using HLA-DR-CD38- CD8 T cells from the same sample as gating controls (filled histogram peaks, “negative” in respect to Ki67 and ICOS, and “positive” regarding Bcl-2). (B–E) Analysis of CD8+ T cells in patient PBMC samples collected before (pre-Dex) and after (post-Dex) administration of dexamethasone. (B) HLA-DRhiCD38hi “effector” lymphocytes as a percentage of total CD8+ T cells. (C–D) Proportion of CD8+ T cells expressing the proliferation marker Ki67+ (C) and activation marker ICOS (D). (E) CD8+ T cell to Treg ratio, calculated by dividing the CD8+ percentage of total T cells by the Treg percentage of total T cells. Each dot represents an individual patient; significant difference between pre-Dex and post-Dex values: ***p < 0.0001, paired students t-test.

Fig 4: Dex treatment increases the proliferation and activation state of Tregs. (A) Representative flow cytometry data, demonstrating the gating strategy used for Treg identification and analysis. Forward scatter (FSC) area vs. FSC-height was used for doublet discrimination, and lymphocytes subsequently selected by FSC vs. side scatter. A “dump” channel was used to gate out dead cells (LIVE/DEAD fixable aqua viability stain), CD14+, CD56+, and CD19+ cells. CD4+ T cells were subsequently selected on the basis of CD4 vs. CD3 staining, followed by the identification of Tregs as CD25hi, CD127lo, and Foxp3+. Tregs, or non-Treg CD4+ T cells, were further gated for expression of Ki67 and ICOS. (B–F) Analysis of Tregs in patient PBMC samples collected before (pre-Dex) and after (post-Dex) administration of Dex. (B) Percentage of Tregs (CD25+CD127loFoxp3+) as a proportion of total CD4+ T cells in PBMC samples. (C–D) Proportion of Tregs (C) and non-Treg CD4+ lymphocytes (D) expressing the proliferation marker Ki67. (E–F) Changes in proportional expression of the activation marker ICOS, in Treg (E) and non-Treg CD4+ lymphocytes (F). Each dot represents an individual patient; significant difference between pre-Dex and post-Dex values: ***p < 0.0001, paired students t-test.

Fig 5: Profile of macrophages after spheroid-induced polarization over time. Spheroids were generated from MCF-7, HT-29, PANC-1, and MIA PaCa-2 tumor cells and 1BR.3.G fibroblasts. CD14+ monocytes were added to 7-day-old spheroids, and 3, 5, and 7 days later, spheroids were analyzed by flow cytometry. The CD11b+ cells were analyzed for surface expression of MHC class II, CD163, CD86, CD206, CD40, and CD14. The CD11b+ cells from the spheroid-polarized MDMs were compared to control M1 and M2a macrophages generated by cytokine cocktails and unstimulated monocytes. Each point represents cells from 5 pooled and dissociated spheroids from each of 4 monocyte donors, except for control M1, M2a macrophages, and unstimulated monocytes. MHC class II expression on day 3 only represents data from 1 experiment with 2 monocyte donors. Statistical tests are performed within the same days. * = p-value of <0.05. ** = p-values of <0.01.